U.S. patent application number 17/285767 was filed with the patent office on 2021-12-16 for neutral multidomain peptide hydrogels and uses thereof.
This patent application is currently assigned to William Marsh Rice University. The applicant listed for this patent is William Marsh Rice University. Invention is credited to Jeffrey HARTGERINK, David LEACH, Tania LOPEZ-SILVA.
Application Number | 20210386907 17/285767 |
Document ID | / |
Family ID | 1000005850910 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210386907 |
Kind Code |
A1 |
HARTGERINK; Jeffrey ; et
al. |
December 16, 2021 |
NEUTRAL MULTIDOMAIN PEPTIDE HYDROGELS AND USES THEREOF
Abstract
Provided herein are peptide-based hydrogels, or neutral
multidomain peptide hydrogel (NMDP), as well as uses thereof. The
uses include encapsulating cells to induce quiescence for long-term
storage and administering to a subject to induce collagen
deposition and macrophage infiltration. The disclosed hydrogel is
useful for the preservation of stem cells, including maintaining
their quiescence and differentiation potential.
Inventors: |
HARTGERINK; Jeffrey;
(Houston, TX) ; LEACH; David; (Houston, TX)
; LOPEZ-SILVA; Tania; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
William Marsh Rice University |
Houston |
TX |
US |
|
|
Assignee: |
William Marsh Rice
University
Houston
TX
|
Family ID: |
1000005850910 |
Appl. No.: |
17/285767 |
Filed: |
October 16, 2019 |
PCT Filed: |
October 16, 2019 |
PCT NO: |
PCT/US2019/056581 |
371 Date: |
April 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62746391 |
Oct 16, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 1/0231 20130101;
C12N 11/04 20130101; A61L 27/22 20130101; A61L 27/52 20130101 |
International
Class: |
A61L 27/22 20060101
A61L027/22; A01N 1/02 20060101 A01N001/02; A61L 27/52 20060101
A61L027/52; C12N 11/04 20060101 C12N011/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. R01 DE021798 awarded by the National Institutes of Health and
under Grant No. DGE1450681 awarded by the National Science
Foundation. The government has certain rights in the invention.
Claims
1. A composition comprising a plurality of peptides; wherein each
peptide of the plurality of peptides comprises a first domain, a
second domain, and a third domain; wherein the first and third
domain are each X.sub.m and m is 1-6; wherein the first domain is
positioned at the N-terminal end of the second domain; wherein the
third domain is positioned at the C-terminal end of the second
domain; and wherein the second domain comprises alternating
hydrophobic (H) and hydrophilic (p) amino acids.
2. The composition of claim 1, wherein the hydrophilic (p) amino
acids are polar amino acids.
3. The composition of claim 1, wherein the hydrophilic (p) amino
acids are selected from the group consisting of S, T, N, and Q.
4. The composition of claim 1, wherein the hydrophobic (H) amino
acids are selected from the group consisting of L, I, V, A, F, Y,
W, and M.
5. The composition of claim 1, wherein the second domain comprises
(Hp).sub.n.
6. The composition of claim 1, wherein the second domain comprises
(pH).sub.n.
7. The composition of claim 5 or 6, wherein n is 4-6.
8. The composition of claim 7, wherein the second domain comprises
(SerLeu).sub.6.
9. The composition of claim 1, wherein X is an amino acid that
lacks hydrogens on the peptide backbone nitrogen.
10. The composition of claim 9, wherein X is selected from the
group consisting of 3'-hydroxyproline, 4'-hydroxyproline, proline,
and an N-modified polar amino acid.
11. The composition of claim 10, wherein the N-modified polar amino
acid is N-methylated or N-alkylated.
12. The composition of claim 10, wherein the N-modified polar amino
acid is N-R-Ser, N-R-Thr, N-R-Asn, or N-R-Gln.
13. The composition of any one of claims 1-12, wherein the peptides
are N-terminally acetylated.
14. The composition of any one of claims 1-13, wherein the peptides
are produced using solid-phase synthesis.
15. The composition of any one of claims 1-14, wherein m is 1.
16. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 1.
17. The composition of any one of claims 1-14, wherein m is 2.
18. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 2.
19. The composition of any one of claims 1-14, wherein m is 3.
20. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 3.
21. The composition of any one of claims 1-14, wherein m is 4.
22. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 4.
23. The composition of any one of claims 1-14, wherein m is 5.
24. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 5.
25. The composition of any one of claims 1-14, wherein m is 6.
26. The composition of any one of claims 1-14, wherein the sequence
of each peptide comprises SEQ ID NO: 6.
27. The composition of any one of claims 1-26, wherein the peptides
further comprise a biologically active peptide mimic.
28. The composition of claim 27, wherein the biologically active
peptide is attached to the N-terminus of the peptides.
29. The composition of claim 27, wherein the biologically active
peptide is attached to the C-terminus of the peptides.
30. The composition of claim 28 or 29, wherein the peptides further
comprise a glycine spacer positioned between the peptide and the
biologically active peptide mimic.
31. The composition of claim 30, wherein the glycine space
comprises between one and three glycine residues.
32. The composition of any one of claims 27-31, wherein the
biologically active peptide mimic has a sequence selected from the
group consisting of one of SED ID NOs: 10-20.
33. The composition of any one of claims 1-32, wherein the
composition is lyophilized.
34. The composition of any one of claims 1-32, wherein the
composition further comprises viable cells.
35. A nanofiber comprising a plurality of peptides according to any
one of claims 1-32.
36. A hydrogel comprising a plurality of peptides according to any
one of claims 1-32.
37. The hydrogel of claim 36, wherein the hydrogel is
biocompatible.
38. The hydrogel of claim 36 or 37, wherein the hydrogel remains
intact at pH 3-11.
39. The hydrogel of any one of claims 36-38, wherein the hydrogel
remains intact at physiological pH.
40. The hydrogel of any one of claims 36-39, wherein the hydrogel
has a storage modulus (G') of less than 100 Pa.
41. The hydrogel of any one of claims 36-40, wherein the hydrogel
has a storage modulus (G') of less than 50 Pa.
42. A method of forming a neutral hydrogel comprising subjecting a
composition of any one of claims 1-32 to ultrasonication at
physiological pH (7.2-7.4).
43. A method for inducing quiescence in cells comprising
encapsulating the cells in a hydrogel of any one of claims
36-41.
44. The method of claim 43, wherein the encapsulated cells remain
viable for at least five days.
45. The method of claim 44, wherein the encapsulated cells remain
viable for at least 30 days.
46. The method of any one of claims 43-45, wherein the method is
further defined as a method for cell preservation.
47. The method of any one of claims 43-46, wherein the cells are
stem cells.
48. The method of claim 47, wherein the stem cells are
hematopoietic stem cells, mesenchymal stem cells, human embryonic
stem cells, or induced-pluripotent stem cells.
49. The method of claim 47, wherein the method maintains the
differential potential of the stem cells.
50. The method of any one of claims 43-49, wherein the cells do not
proliferate once encapsulated in the hydrogel.
51. The method of any one of claims 49-50, wherein the cells do not
attach to the hydrogel.
52. A method comprising administering a hydrogel as provided in any
one of claims 36-41 to a target location in a subject.
53. The method of claim 52, wherein administering comprises
injecting.
54. The method of claim 52 or 53, wherein the subject is a
mammal.
55. The method of claim 54, wherein the subject is a human patient,
a mouse, a rat, or a pig.
56. The method of any one of claims 52-55, wherein the target
location is a site comprising damaged tissue.
57. The method of any one of claims 52-56, wherein the target
location is a site in need of collagen deposition.
58. The method of claim 57, wherein the method is further defined
as a method for stimulating collagen deposition at a target
location in a patient in need thereof.
59. The method of any one of claims 52-58, wherein the method
causes macrophages or any immune cell to infiltrate the target
location.
60. The method of any one of claims 52-59, wherein the
concentration of peptides in the administered hydrogel is about 3-7
mM.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority benefit of U.S.
provisional application No. 62/746,391, filed Oct. 16, 2018, the
entire contents of which is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing, which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 1, 2019, is named RICEP0055WO_ST25.txt and is 5.7 kilobytes
in size.
BACKGROUND
[0004] The development of this disclosure was funded in part by the
Welch Foundation under Grant No. C-1557.
1. Field
[0005] The present invention relates generally to the fields of
chemistry, cell biology, and medicine. More particularly, it
concerns neutral multidomain peptide hydrogels as well as methods
of use thereof.
2. Description of Related Art
[0006] Of great promise for regenerative medicine and diverse
medical problems is the use of stem cells in clinical therapy. Stem
cells have the capacity to differentiate into a great variety of
tissues and have been shown to be effective in the treatment of
autoimmune diseases, leukemia, and myocardial infarction. Despite
the potential of stem cells, their therapeutic use is challenging
because they can lose their potency and differentiation capacity
during manipulation in vitro after isolation. In addition, the
preservation of stem cells is critical for manufacturing and
clinical use because it allows for the development of cell banks,
the performance of quality and safety testing, transportation,
long-term storage, processing, and better availability. The current
methods for stem cell preservation are cryopreservation,
freeze-drying, and quiescence maintenance in culture.
Cryopreservation requires the use of cryopreservatives, such as
DMSO, ethylenglycol, serum, and additives. On the other hand,
short-term quiescence maintenance in vitro requires the use of
specific medium containing a diversity of growth factors and
biological cues that need to be optimized for each cell type.
However, there is no universal set of conditions for preserving
stem cells, and the preservation processes, preservatives, and
additives used (such as DMSO) can have adverse effects that make
cell recovery and protection of cell activity challenging. The
development of a simple and effective preservation method for
quiescent stem cells would facilitate the translation of stem cell
therapies to the clinic. Therefore, compositions and methods for
maintaining stem cells in quiescence, a reversible inactive state
demonstrating minimal basal cell activity, are needed to allow for
the preservation of these cells for prolonged periods of time.
SUMMARY
[0007] Provided here are peptide-based hydrogels, or neutral
multidomain peptide hydrogel (NMDP) as well as uses thereof. When
NMDP is used as a scaffold for cell encapsulation, it induces a
quiescence state in cells and promotes their preservation while
maintaining their viability and growing capacity over time. The
chemical and physical properties of this material make it useful
for the preservation of stem cells by maintaining their quiescence
and differentiation potential.
[0008] In one embodiment, provided herein are compositions
comprising a plurality of peptides; wherein each peptide of the
plurality of peptides comprises a first domain, a second domain,
and a third domain; wherein the first and third domain are each
X.sub.m and m is 1-6; wherein the first domain is positioned at the
N-terminal end of the second domain; wherein the third domain is
positioned at the C-terminal end of the second domain; and wherein
the second domain comprises alternating hydrophobic (H) and
hydrophilic (p) amino acids.
[0009] In some aspects, the hydrophilic (p) amino acids are polar
amino acids. In some aspects, the hydrophilic (p) amino acids are
selected from the group consisting of S, T, N, and Q. In some
aspects, the hydrophobic (H) amino acids are selected from the
group consisting of L, I, V, A, F, Y, W, and M. In some aspects,
the second domain comprises (Hp).sub.n. In some aspects, the second
domain comprises (pH).sub.n. N may be 4-6. In some aspects, the
second domain comprises (SerLeu).sub.6.
[0010] In some aspects, X is an amino acid that lacks hydrogens on
the peptide backbone nitrogen. In some aspects, X has no potential
to form beta-sheet interactions in the NMDP peptide termini. In
some aspects, X is selected from the group consisting of
3'-hydroxyproline, 4'-hydroxyproline, proline, and an N-modified
polar amino acid. In certain aspects, the N-modified polar amino
acid is N-methylated or N-alkylated. In certain aspects, the
N-modified polar amino acid is N-R-Ser, N-R-Thr, N-R-Asn, or
N-R-Gln, such as, for example, those shown in Scheme 1.
##STR00001## ##STR00002##
[0011] In some aspects, the peptides are N-terminally acetylated.
In some aspects, the peptides are produced using solid-phase
synthesis. In some aspects, m is 1, 2, 3, 4, 5, or 6. In some
aspects, the sequence of each peptide comprises any one of SEQ ID
NOs: 1-6.
[0012] In some aspects, the peptides further comprise a
biologically active peptide mimic. In certain aspects, the
biologically active peptide is attached to the N-terminus of the
peptides. In certain aspects, the biologically active peptide is
attached to the C-terminus of the peptides. In some aspects, the
peptides further comprise a glycine spacer positioned between the
peptide and the biologically active peptide mimic. In certain
aspects, the glycine space comprises between one and three glycine
residues. In certain aspects, the biologically active peptide mimic
has a sequence selected from the group consisting of one of SED ID
NOs: 10-20. In certain aspects, the biologically active peptide
mimic is selected from those presented in Table 1.
TABLE-US-00001 TABLE 1 Biologically active peptide mimics Peptide
SEQ mimic ID sequence NO: Origin Bioactivity RGDS 10 Fibronectin
Cell attachment IKVAV 11 Laminin Neurite outgrowth KDI 12
Laminin-gamma 1 Neurite outgrowth RNIAEIIKDI 13 Laminin-gamma 1
Neurite outgrowth VFDNFVLK 14 Tenascin-C Neurite outgrowth DWIVA 15
BMP-2 Bone formation AQFHRHKQLIR 16 IL-4 Anti-inflammatory FLKRA
RELRYLRRA 17 IL-4 Anti-inflammatory FLPASGL 18 TGF-.beta.1
Inflammation ESPLKRQ 19 TGF-.beta.1 Inflammation DPHIKLQLQAE 20
FGF-2 Wound healing
[0013] In some aspects, the composition is lyophilized. In some
aspects, the compositions further comprise viable cells.
[0014] In one embodiment, provided herein is a nanofiber comprising
a plurality of peptides according to any one of the present
embodiments. In one embodiment, provided herein are hydrogels
comprising a plurality of peptides according to any one of the
present embodiments. In some aspects, the hydrogel is
biocompatible. In some aspects, the hydrogel remains intact at pH
3-11. In some aspects, the hydrogel remains intact at physiological
pH. In some aspects, the hydrogel has a storage modulus (G') of
less than 100 Pa. In some aspects, the hydrogel has a storage
modulus (G') of less than 50 Pa. In one embodiment, provided herein
are methods of forming a neutral hydrogel comprising subjecting a
composition of any one of the present embodiments to
ultrasonication at physiological pH (7.2-7.4).
[0015] In one embodiment, provided herein are methods for inducing
quiescence in cells comprising encapsulating the cells in a
hydrogel of any one of the present embodiments. In some aspects,
the encapsulated cells remain viable for at least five days. In
some aspects, the encapsulated cells remain viable for at least 30
days. In some aspects, the methods are further defined as methods
for cell preservation.
[0016] In some aspects, the cells are stem cells. In certain
aspects, the stem cells are hematopoietic stem cells, mesenchymal
stem cells, human embryonic stem cells, or induced-pluripotent stem
cells. In some aspects, the methods maintain the differential
potential of the stem cells. In some aspects, the cells do not
proliferate once encapsulated in the hydrogel. In some aspects, the
cells do not attach to the hydrogel.
[0017] In one embodiment, provided herein are methods comprising
administering a hydrogel as provided in any one of the present
embodiments to a target location in a subject. In some aspects,
administering comprises injecting. In some aspects, the subject is
a mammal. In some aspects, the subject is a human patient, a mouse,
a rat, or a pig. In some aspects, the target location is a site
comprising damaged tissue. In some aspects, the target location is
a site in need of collagen deposition. In some aspects, the methods
are further defined as methods for stimulating collagen deposition
at a target location in a patient in need thereof. In some aspects,
the methods cause macrophages to infiltrate the target location. In
some aspects, the concentration of peptides in the administered
hydrogel is about 3-7 mM.
[0018] As used herein, "essentially free," in terms of a specified
component, is used herein to mean that none of the specified
component has been purposefully formulated into a composition
and/or is present only as a contaminant or in trace amounts. The
total amount of the specified component resulting from any
unintended contamination of a composition is therefore well below
0.05%, preferably below 0.01%. Most preferred is a composition in
which no amount of the specified component can be detected with
standard analytical methods.
[0019] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising," the words "a" or "an" may mean one or
more than one.
[0020] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0021] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the inherent variation in the method being employed to
determine the value, the variation that exists among the study
subjects, or a value plus or minus 5% of the stated number.
[0022] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0024] FIG. 1. Rational design of O.sub.n(SL).sub.6O.sub.n peptide
series. 4-Hydroxyproline (O) domains of variable length are
incorporated into the N and C termini to create a bulky non-ionic
domain. In solution, the amphiphilic domain has an antiparallel
.beta.-sheet conformation and assembles by H-bonding between
peptide backbones. The non-ionic domain provokes steric
interactions that frustrate the infinite aggregation of the MDPs
forming nanofibers.
[0025] FIGS. 2A-C. Secondary structure and nanostructure
characterization of O.sub.n(SL).sub.6O.sub.n peptide series. FIG.
2A: ATR FTIR spectra of the amide I and amide II region of dried
peptide samples. All peptides have characteristic peaks for
antiparallel .beta.-sheet structure. FIG. 2B: CD spectra of 1 wt. %
(O.sub.1 [top solid line at 195 nm]: 7 mM, O.sub.2 [top dashed line
at 195 nm]: 6 mM, O.sub.3 [middle solid line at 195 nm] and O.sub.4
[middle dashed line at 195 nm]: 5 mM, O.sub.5 [bottom solid line at
195 nm] and O.sub.6 [bottom dashed line at 195 nm]: 4 mM) peptide
solutions in TFE at 25.degree. C. FIG. 2C: Negative-stained TEM
images in water. O.sub.1, O.sub.2, and O.sub.5 at 0.01 wt. %,
O.sub.3, O.sub.4 and O.sub.6 at 0.02 wt. %. Scale bar=100 nm.
[0026] FIG. 3. O.sub.5(SL).sub.6O.sub.5 hydrogel properties are pH
and ionic strength independent. CD spectra of the positively
charged MDP, K.sub.2(SL).sub.6K.sub.2, neutral O.sub.5 and
negatively charged, E.sub.2(SL).sub.6E.sub.2 at different pH.
K.sub.2 and E.sub.2 hydrogel formation is dependent on pH, whereas
O.sub.5 remains a hydrogel at acidic, neutral and basic pH. For
K.sub.2, at 200 nm, the top line is basic, the middle line is
acidic, and the bottom line is neutral. For O.sub.5, at 210 nm, the
top line is basic, the middle line is neutral, and the bottom line
is acidic. For E.sub.2, at 190 nm, the top line is neutral, the
middle line is basic, and the bottom line is acidic.
[0027] FIGS. 4A-D. O.sub.5(SL).sub.6O.sub.5 peptide solution forms
a hydrogel upon treatment with ultrasonication. FIG. 4A: Structural
characterization of 1 wt. % peptide solutions in 149 mM sucrose and
0.5.times.HBSS. FIG. 4B: SEM micrograph of 1 wt. % O.sub.5 hydrogel
showing an entangled fibrillar structure. Scale bar=1 .mu.m. FIGS.
4C-D: Strain sweep (FIG. 4C) and shear recovery (FIG. 4D) of 1 wt.
% O.sub.5 peptide hydrogel.
[0028] FIGS. 5A-E. 3-D culture of NIH-3T3 fibroblasts in 1 wt. %
O.sub.5 hydrogel and the positively charged peptide hydrogel
K.sub.2(SL).sub.6K.sub.2. FIG. 5A: Cell viability of NIH-3T3
fibroblasts at day 1, 3 and 5 by Calcein AM and Ethidium
Homodimer-1 staining (viable cells in green, dead cells in red).
Scale bar=100 .mu.m. FIGS. 5B-C: Actin cytoskeleton staining of
NIH-3T3 fibroblasts encapsulated for 5 days in K.sub.2 (FIG. 5B)
and O.sub.5 (FIG. 5C). AlexaFluor 488-phalloidin (green) and DAPI
(blue). Scale bar=50 .mu.m. FIG. 5D: Percentage of viable cells
present in K.sub.2 and O.sub.5 at day 1, day 3, and day 5 after
seeding. FIG. 5E: Viable cell density (cells/mm.sup.3) at day 1,
day 3, and day 5. Error bars represent standard deviation
(n=6).
[0029] FIG. 6. MALDI TOF MS of O.sub.6(SL).sub.6O.sub.n series.
[0030] FIG. 7. Basis spectra for CD spectroscopy data fitting in a)
2,2,2-trifluoroethanol (TFE) 1 wt. % solutions. At 200 nm, the top
line is O.sub.1-.beta.-sheet, the middle line is
K.sub.2-.alpha.-helix, and the bottom line is
(Hyp).sub.6-Polyproline type II.
[0031] FIG. 8. Experimental and fit CD spectra of the O peptide
series calculated by the least square linear method. At 210 nm, the
top lines are Experimental and the bottom lines are Fit.
[0032] FIG. 9. Negative-stained TEM images of
O.sub.n(SL).sub.6O.sub.n dissolved initially in
2,2,2-trifluoroethanol (TFE) at 1 wt. % and diluted with water to
final concentration. O.sub.1 at 0.001 wt. %, O.sub.2, O.sub.3,
O.sub.4, O.sub.5 and O.sub.6 at 0.01 wt. %. Scale bar=100 nm.
[0033] FIG. 10. Viscoelastic properties of O.sub.5(SL).sub.6O.sub.5
peptide hydrogel at 1 wt. % in PBS (5 mM phosphate and 150 mM
NaCl).
[0034] FIG. 11. Inverted-vial test of K.sub.2(SL).sub.6K.sub.2,
O.sub.5(SL).sub.6O.sub.5 and E.sub.2(SL).sub.6E.sub.2 peptide
solutions/hydrogel at acidic, neutral and basic pH. Positively
charged MPD (K.sub.2) forms a gel at pH 11, negatively charged MDP
(E.sub.2) forms a gel at acidic pH and undergoes phase separation.
Neutral MDP (O.sub.5) remains a hydrogel at all pH and without the
presence of multivalent ions.
[0035] FIG. 12. Frequency sweep of O.sub.5(SL).sub.6O.sub.5 peptide
hydrogel at 1 wt. % in 149 mM sucrose and 0.5.times.HBSS.
[0036] FIG. 13. NIH-3T3 fibroblasts cultured in 1 wt. % O.sub.5 for
5 days grow and proliferate again after harvesting from the peptide
scaffold. 30 min after harvesting, the cells are still in the
balled-up morphology, but they start adhering to the tissue plastic
and recover.
[0037] FIGS. 14A-C. H&E and immunofluorescent staining of
subcutaneous implants of O.sub.5 hydrogel. FIG. 14A shows day 3,
FIG. 14B shows day 7, and FIG. 14C shows day 14. After
implantation, the hydrogel is infiltrated by macrophages (F4/80+
cells: dark gray, nuclei: light gray), which degrade the material
and remodel the native tissue. By day 14, the implant size
decreases and new collagen is observed similar to native tissue
(FIG. 16C). Scale bar 100 .mu.m. Dotted line indicates borders of
the hydrogel implant.
[0038] FIGS. 15A-C. H&E staining of O.sub.5(SL).sub.6O.sub.5
implants at day 3 (FIG. 15A), 7 (FIG. 15B), and 14 (FIG. 15C) after
injection in a subcutaneous mouse model. Implant is degraded over
time and size decreases. Scale bar: 1 mm.
[0039] FIGS. 16A-C. Masson's Trichome of O.sub.5(SL).sub.6O.sub.5
implants at day 3 (FIG. 16A), 7 (FIG. 16B), and 14 (FIG. 16C) after
injection in a subcutaneous mouse model. The material is degraded
and replaced by collagen similar to native tissue. Scale bar: 100
.mu.m.
DETAILED DESCRIPTION
[0040] A simple and effective method to preserve quiescent stem
cells is necessary to allow the use of these cells for different
medical applications. The methods provided herein will facilitate
the storage and availability of stem cells (e.g., hematopoietic
stem cells, mesenchymal stem cells, human embryonic stem cells,
induced-pluripotent stem cells) for long-term use, promote the
development of cell banks, and allow for the performance of quality
and safety testing, cell transportation, and processing.
[0041] The self-assembly of multidomain peptides need not involve
the use of charged amino acids. Uncharged domains of
oligo-hydroxyproline at the peptide termini form bulky helical
structures that create molecular frustration between the
.beta.-sheet self-assembling domain and terminal steric
interactions. These steric interactions from the hydroxyproline
domains affect peptide solubility, aggregation, and nanofiber
structure. While all peptides in the O.sub.n(SL).sub.6O.sub.n
peptide series (n=1-6) formed nanofibers, only
O.sub.5(SL).sub.6O.sub.5 was able to form a hydrogel. This
nanostructured hydrogel shows promising properties for biomedical
applications, including rapid shear recovery and support for cell
viability. It also appears to induce a quiescent state in cells
over time, a property that may be desirable for cell preservation.
This compliant hydrogel is promising as a cell preservation method,
where the cells can be 3D encapsulated in this matrix with the
required trophic support and stored for longer times. The cells can
be released from the nanofibrous material by simply dispersing in
media, recovering, and using for their final purpose. When
implanted in vivo, the hydrogel is infiltrated by macrophages
without evidence of eliciting an adverse inflammatory response and
the implant is degraded over time. This work establishes the
creation of a neutral biocompatible hydrogel and extends the scope
of peptide-based self-assembling materials to encompass positive,
negative, and neutral nanofiber chemistries for diverse biomaterial
designs.
I. SELF-ASSEMBLING PEPTIDE HYDROGELS
[0042] Self-assembling peptides have been extensively studied
because of their relationship to protein folding and assembly.
These peptides can be designed to spontaneously self-assemble into
fibrous nanomaterials that have a chemical composition and
nanostructure that is readily interfaced with living systems and
possess material properties that mimic the extracellular matrix
(Lutolf & Hubbell, 2005). The resulting bio-interactive nature
of these supramolecular materials is useful in biomedical
applications, such as controlled drug and protein delivery, cell
encapsulation, and tissue regeneration (Webber et al., 2015;
Rad-Malekshahi et al., 2016; Leach et al., 2018). Several peptide
designs have been created to self-assemble into nanoribbons,
monolayers or nanofibers, which utilize amphiphilic and ionic
domains to govern their supramolecular structure (Aggeli et al.,
2003; Powers et al., 2002; Holmes et al., 2000; Cormier et al.,
2013; Pochan et al., 2003; Hargerink et al., 2001).
[0043] Generally, self-assembling peptide design has required the
incorporation of charged amino acids into the peptide sequence as a
mechanism to control assembly and material properties by stimuli
such as pH and ionic strength (Chockalingam et al., 2007;
Micklitsch et al., 2011; Schneider et al., 2002). However, it is
known that biological responses and cell behavior highly depend on
the chemistry and charge of the interacting materials. For example,
poly-lysine is commonly used for cell attachment and proliferation
in culture techniques (Mazie et al., 1975; Blau, 2013), but this
and many other cationic polymers show an inherent
concentration-dependent cytotoxicity (Fischer et al., 2003;
Kadlecova et al., 2012). On the other hand, neutral polymers, such
as PEG, are inert, biocompatible, and non-immunogenic and have been
used in countless studies to bestow these "stealth" properties on
other materials (Zhu, 2010; Meyers & Grinstaff, 2012; Li et
al., 2015). Neutral, non-ionic peptides tend to have poor
solubility and aggregate or precipitate in aqueous solutions. This
makes controlling the formation of finite supramolecular structures
derived from these molecules challenging. Previously, steric
interactions and size mismatch have been used to regulate the
self-assembly of polymeric mushroom-shaped nanostructures (Stupp et
al., 1997). Nevertheless, the field is limited by its ability to
explore biological responses to only positively or negatively
charged self-assembling peptide hydrogels because strategies to
control peptide self-assembly through uncharged residues are
limited.
[0044] Three-dimensional cell encapsulation in hydrogels have been
use as a culture method, where cells have the support and
biological cues to grow and proliferate in the material scaffold.
These hydrogels are designed to promote cell attachment, growth and
proliferation, while the hydrogel is being degraded over time.
However, for stem cell preservation it is required to maintain the
cell quiescence and avoid any potential differentiation until the
cells are required. The use of hydrogels for stem cell preservation
would provide a simple and less harmful method for cell storage.
For that purpose, the material should not promote cell attachment
or exogenous cues for the cells to proliferate, it should induce
and maintain the quiescence state of stem cells, while still
protecting cell viability for long periods of time.
[0045] Herein, steric frustration was used as a controlling
mechanism for the solubility and self-assembly of neutral peptides.
A series of peptides were designed to determine if self-assembly
could be mediated by the steric impediment created by neutral
hydroxyproline domains, eliminating the need for high charge. The
absence of charged groups allowed the assembly and structure of the
peptide to remain largely independent of pH and ionic strength.
Peptide solubility and nanofiber length increased with a higher
number of hydroxyproline residues giving O.sub.5(SL).sub.6O.sub.5
the optimal properties for self-assembly and hydrogelation. In
vitro this hydrogel supports cell viability, while in vivo it is
infiltrated with cells and easily degraded over time without
promoting a strong inflammatory response. This neutral
self-assembling peptide hydrogel shows promising properties for
biomedical and cell preservation applications and expands the scope
of available self-assembling peptide nanostructured materials that
may be studied for applications in biology and medicine.
[0046] The present neutral, self-assembling multidomain peptide
(NMDP) was designed to form a cytocompatible hydrogel material at
physiological pH upon ultrasonication treatment. Gelation is
independent of the presence of ions and pH variations, making this
material robust and simple to use for cell culture. The NMDP
hydrogel's viscoelastic properties, storage modulus (23.3.+-.8.7
Pa), and loss modulus (1.7.+-.0.5 Pa) demonstrate that this peptide
forms a self-supporting compliant flexible gel, which can be easily
pipetted or injected because of its shear thinning and shear
recovery characteristics. The NMDP hydrogel exhibits a liquid
behavior when high strain is applied, while having a very rapid
recovery of approximately 92% of its initial storage modulus 1
minute after the strain is released.
[0047] The production of the present NMDP can follow these steps:
Step 1: The peptide is synthesized using solid-phase peptide
synthesis and FMOC strategy. The N-termini are acetylated before
cleavage with TFA and scavengers. TFA is removed by rotary
evaporation and solid peptide is recovered by trituration with
diethyl ether. The NMDP is dialyzed against MQ H.sub.2O in 100-500
Da, pH adjusted to 7.2-7.4, sterile filtered, lyophilized, and
stored at -20.degree. C.
[0048] Step 2: Peptide solution of NMDP is prepared at 1% by weight
in 149 mM sucrose and 0.5.times.HBSS. Then, the peptide solution is
sonicated using a Microson Ultrasonic Cell Disruptor with a 2 mm
microprobe at room temperature for 1 or 10 cycles, where each cycle
consisted of 10 pulses and 1 min relaxation time between cycles.
Sonication must be done before any cells are mixed. NMDP forms a
compliant material that must be handled with more precautions.
Changes of preservation media or buffer must be done with careful
pipetting, otherwise there would be cell and material losses.
[0049] Step 3: Cells are isolated and incubated at 37.degree. C.
and 5% CO.sub.2 before encapsulation. Cells are centrifuged for 6
min at 1600 rpm, then the cell pellet is dispersed in HBSS, counted
and adjusted to the required concentration. The neutral hydrogel is
prepared by sonication, as previously described, using only 90% of
the required 149 mM sucrose-0.5.times.HBSS to obtain a 1% by weight
concentration, the remaining 10% was added as a cell suspension,
mixed, and homogenized. Conditions for the cell medium may be
optimized for each cell type.
[0050] NIH 3T3 fibroblasts were encapsulated in NMDP hydrogel and
cultured for up to 5 days. To evaluate the cytocompatibility and
cell behavior promoted by NDMP, a cell viability test was performed
using Calcein AM and Ethidium homodimer-1. Fibroblasts encapsulated
in the NMDP hydrogel showed good initial viability, which remained
constant throughout the experiment. Despite high and consistent
cell viability in NMDP over the course of this study, the
encapsulated fibroblasts were not observed to proliferate. The
cells did not spread and adhere to the material, instead they
remained "balled up." The lack of attachment to the hydrogel and
balled up morphology suggest that the NMDP has achieved a "stealth"
behavior present in other neutral polymers, such as PEG. All these
observations suggest that fibroblasts cultured in NMDP are in a
quiescent state. The ability of NMDP hydrogel to keep cells alive
without proliferation is a promising result with potential
applications for the use of this material in cell preservation.
[0051] The use of this novel NMDP for cell preservation avoids the
use of exogenous factors to promote cell quiescence and the harsh
conditions required for cryopreservation and freeze-thaw processes.
The chemical design and viscoelastic properties of this neutral
self-assembling peptide hydrogel provides a favorable environment
for the cells to go into an inactive state while maintaining their
viability and phenotype. The cells can be easily released from the
hydrogel by simple disturbing the peptide matrix and resuspending
the cells in media or buffer.
[0052] The peptides of the present disclosure can be lyophilized
and dissolved in, for example, an appropriate concentration of
sucrose solution or in deionized water. In one embodiment, the
peptides can be provided in a 1-300 mM sucrose solution. The
peptide concentration in the solution may be from about 0.1 mg/ml
to about 100 mg/ml, from about 1 mg/ml to about 90 mg/ml, from
about 10 mg/ml to about 80 mg/ml, from about 20 mg/ml to about 70
mg/ml, from about 30 mg/ml to about 60 mg/ml, from about 40 mg/ml
to about 50 mg/ml, and any concentration therebetween.
II. PHARMACEUTICAL FORMULATIONS
[0053] The provided neutral multidomain peptides can be combined
with a pharmaceutically acceptable carrier or vehicle for
administration to human or animal subjects. In some embodiments,
more than one multidomain peptide or peptide analog can be combined
to form a single preparation. The multidomain peptides or peptide
analogs can be conveniently presented in unit dosage form and
prepared using conventional pharmaceutical techniques. Such
techniques include the step of bringing into association the active
ingredient and the pharmaceutical carrier(s) or excipient(s). In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers. Formulations suitable for parenteral administration
include aqueous and nonaqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampules and vials, and may be stored in a freeze-dried
(lyophilized) condition.
[0054] In certain, embodiments, unit dosage formulations are those
containing a dose or unit, or an appropriate fraction thereof, of
the administered ingredient. It should be understood that in
addition to the ingredients particularly mentioned above,
formulations encompassed herein may include other agents commonly
used by one of ordinary skill in the art.
[0055] The pharmaceutical compositions provided herein may be
administered through different routes, such as parenteral,
intraperitoneal, intramuscular, subcutaneous, and intratumoral. It
may be desirable to administer the pharmaceutical compositions
locally to the area in need of treatment. This may be achieved by,
for example, and not by way of limitation, local or regional
infusion or perfusion during surgery, injection, or implant. In one
embodiment, administration can be by direct injection at the site
(or former site) of a tissue that is to be treated.
[0056] The amount of the pharmaceutical compositions that will be
effective depends on the nature of the disorder or condition to be
treated, as well as the stage of the disorder or condition.
Effective amounts can be determined by standard clinical
techniques. The precise dose to be employed in the formulation will
also depend on the route of administration, and should be decided
according to the judgment of the health care practitioner and each
subject's circumstances.
[0057] The specific dose level and frequency of dosage for any
particular subject may be varied and will depend upon a variety of
factors, including the activity of the specific compound, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity
of the condition of the subject undergoing therapy.
[0058] The pharmaceutical compositions of the present disclosure
can be administered at about the same dose throughout a treatment
period, in an escalating dose regimen, or in a loading-dose regime
(e.g., in which the loading dose is about two to five times the
maintenance dose). In some embodiments, the dose is varied during
the course of a treatment based on the condition of the subject
being treated, the severity of the disease or condition, the
apparent response to the therapy, and/or other factors as judged by
one of ordinary skill in the art. The volume of administration will
vary depending on the route of administration. Those of ordinary
skill in the art will know appropriate volumes for different routes
of administration.
III. METHODS OF TREATMENT
[0059] The present invention provides methods of treating a patient
with neutral multidomain peptide hydrogels as provided herein.
[0060] The term "subject" or "patient" as used herein refers to any
individual to which the subject methods are performed. Generally,
the patient is human, although as will be appreciated by those in
the art, the patient may be an animal Thus other animals, including
mammals such as rodents (including mice, rats, hamsters and guinea
pigs), cats, dogs, rabbits, farm animals including cows, horses,
goats, sheep, pigs, etc., and primates (including monkeys,
chimpanzees, orangutans and gorillas) are included within the
definition of patient.
[0061] "Treatment" and "treating" refer to administration or
application of a therapeutic agent to a subject or performance of a
procedure or modality on a subject for the purpose of obtaining a
therapeutic benefit of a disease or health-related condition.
[0062] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well-being of the subject with
respect to the medical treatment of this condition. This includes,
but is not limited to, a reduction in the frequency or severity of
the signs or symptoms of a disease.
[0063] Likewise, an effective response of a patient or a patient's
"responsiveness" to treatment refers to the clinical or therapeutic
benefit imparted to a patient at risk for, or suffering from, a
disease or disorder. Such benefit may include cellular or
biological responses, a complete response, a partial response, a
stable disease (without progression or relapse), or a response with
a later relapse.
[0064] For the treatment of disease, the appropriate dosage of a
therapeutic composition will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
previous therapy, the patient's clinical history and response to
the agent, and the discretion of the physician. The agent may be
suitably administered to the patient at one time or over a series
of treatments.
IV. KITS
[0065] Kits are envisioned containing peptides or hydrogels of the
present invention. The kit may comprise reagents required for the
formation of or the delivery of the hydrogel. The kit may also
comprise a suitable container means, which is a container that will
not react with components of the kit, such as an eppendorf tube, a
syringe, a bottle, or a tube. The container may be made from
sterilizable materials such as plastic or glass. The kit may
further include an instruction sheet that outlines the procedural
steps of the methods, such as the same procedures as described
herein or are otherwise known to those of ordinary skill.
V. EXAMPLES
[0066] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Materials and Methods
[0067] Peptide synthesis. All peptides were synthesized using
solid-phase peptide synthesis according to the methodology
previously reported (Aulisa et al., 2009). Briefly, a semi-manual
synthesis was accomplish using an Apex Focus XC (Aapptec)
synthesizer for mixing, solvent addition, and wash steps.
Low-loading Rink Amide MBHA resin (0.32 mmol/g) was used, and the
N-termini were acetylated before cleavage with TFA and scavengers.
TFA was removed by rotary evaporation and solid peptide was
recovered by trituration with diethyl ether. O.sub.1 and O.sub.2
were washed with Milli-Q deionized H.sub.2O because of their poor
solubility, while O.sub.3-O.sub.6 were dialyzed against MQ H.sub.2O
for 3-4 days in 100-500 Da or 1000 Da MWCO dialysis tubing
(Spectra/Por, Spectrum Laboratories Inc., Rancho Dominguez,
Calif.). Peptide solutions were adjusted to pH 7.2, sterile
filtered when solubility allowed, frozen, lyophilized, and stored
at -20.degree. C. All peptides were characterized using Autoflex
MALDI-TOF MS (Bruker Instruments, Billerica, Mass.) to confirm
purity and correct mass (FIG. 6). Samples for MALDI-TOF MS
characterization were prepared as follows: peptides were dissolve
in TFE:H.sub.2O:MeOH (1:1:0.2) acidified with 0.05% trifluoracetic
acid (TFA). The peptide solution was mixed with .alpha.-CHCA matrix
solution (in 50% acetonitrile) in a 1 to 1 ratio while being
spotted on a ground steel MALDI target plate. Droplets were allowed
to dry before the analysis.
[0068] Circular Dichroism (CD) spectroscopy. For secondary
structure characterization, peptide solutions of 1% by weight
(molar concentration is included in Table 2) were prepared in
2,2,2-trifluoroethanol (TFE, Sigma Aldrich, St. Louis, Mo.). CD
spectra acquisition was performed in a CD Jasco J-810
spectropolarimeter (Jasco Inc., Easton, Md.) using a 0.01 mm
cuvette. Data were collected at room temperature from 180 to 250 nm
at a speed of 50 nm/min, with a 0.1 nm data pitch and the spectra
were averaged over 5 scans. To study the hydrogelation of O.sub.5,
1% by weight peptide solutions were made in 149 mM sucrose solution
in 0.5.times. Hank's Balanced Salt Solution (HBSS, Life
Technologies) and analyzed as previously described.
[0069] To estimate the secondary structure of O peptide series,
data fitting using the least square linear method was performed in
MATLAB (Mathworks, Natick, Mass.). (Hyp).sub.6,
O.sub.1(SL).sub.6O.sub.1 and K.sub.2(SL).sub.6K.sub.2 were used as
basis spectra for polyproline type II, .beta.-sheet and
.alpha.-helix, respectively (FIG. 7). As has been demonstrated in
other systems, TFE promotes the formation of .alpha.-helical
structures (Myers et al., 1998); therefore, .alpha.-helix was
considered as a possible structure to achieve a better
estimation.
[0070] Attenuated Total Reflectance Fourier Transform Infrared
Spectroscopy (ATR-FTIR). Peptide samples (10 .mu.L of 1% by weight
peptide solution) were dried under nitrogen flow on a Golden Gate
diamond window of an ATR stage or on a glass slide. IR spectra were
collected on a Jasco FT/IR-660 plus spectrometer (Jasco Inc.,
Easton, Md.) and a Nicolet iS5 FT/IR spectrometer (Thermo
Scientific, Waltham, Mass.) at 1 cm.sup.-1 resolution with 64 scans
accumulation, and the background was subtracted, and vapor
reduction was applied to minimize the contribution of water in the
resulting spectra. Spectragryph--optical spectroscopy software was
used for data processing and normalization (Menges, 2017).
[0071] Transmission Electron Microscopy (TEM). For TEM, 1% by
weight peptide solutions in TFE or Milli-Q H.sub.2O were diluted to
concentrations ranging from 0.1% by weight to 0.01% by weight with
Milli-Q water. All samples were prepared via negative staining on
Quantifoil R1.2/1.3 holey carbon films on copper mesh grids. Each
peptide was spotted and allowed to adsorb for 1 min before excess
peptide was blotted. A 2 wt. % phosphotungstic acid (PTA) solution
in Milli-Q water was prepared at pH 7 and sterile filtered. The
sample grids were negative stained for 5 min in PTA. Excess PTA was
wicked off the grids before the samples were dried and imaged at
120 kV and 40K magnification using a JEOL 2010 TEM microscope (JEOL
USA Inc., Peabody, Mass.).
[0072] Hydrogel Formation. Peptide solutions of O.sub.5 were
prepared at 1% by weight (4.2 mM) in 149 mM sucrose and
0.5.times.HBSS. This buffer system was chosen to be consistent with
prior MDP experiments and find optimum gelling conditions for
future in vivo and in vitro work. Peptide solutions were sonicated
using a Microson Ultrasonic Cell Disruptor with a 2 mm microprobe.
The instrument frequency was 22.5 kHz and a power output up to 10
watts (RMS) was used. Solutions were sonicated at room temperature
for 1 or 10 cycles, where each cycle consisted of 10 pulses and 1
min relaxation time between cycles. For the study of hydrogelation
of O.sub.5, K.sub.2 and E.sub.2 at different pH values and ionic
strength, 1% by weight peptide solutions were prepared in deionized
water, PBS, or HBSS. pH was adjusted using HCl or NaOH 0.1 M
solutions.
[0073] Oscillatory Rheology. Rheological properties of O.sub.5
hydrogel were analyzed by oscillatory rheology using a TA
Instruments AR-G2 rheometer (TA Instruments, New Castle, Del.).
Hydrogels were prepared 24 h before the analysis as described
above. 150 .mu.L of 1% by weight peptide hydrogels were transferred
from a cut syringe onto the rheometer stage equipped with a 12 mm
stainless-steel parallel plate set to a 1000 .mu.m gap height. The
following program was used to monitor the storage modulus (G') and
loss modulus (G'') under various conditions as has been previously
published (Li et al., 2017). Strain sweep analysis was performed
using an applied strain of 0.01%-200% at a frequency of 1 rad/s.
Frequency sweep analysis was performed under 1% strain at 0.1-100
rad/s. Shear recovery analysis was performed by subjecting the
peptide sample to 1% strain for 20 min, then 200% for 1 min, and
finally 1% for 20 min, allowing for disruption of the hydrogel and
monitoring of G' and G'' recovery.
[0074] Scanning Electron Microscopy (SEM). For SEM
characterization, 1% by weight peptide hydrogel was prepared as
previously described. Hydrogel was dehydrated with a series of
ethanol from 30% to 100% and the ethanol was removed by critical
point drying using an EMS 850 critical point dryer (Electron
Microscopy Sciences, Hatfield, Pa.). Dried samples were mounted
into SEM pucks with conductive carbon tape and coated with 4 nm of
gold using a Denton Desk V Sputter system (Denton Vacuum,
Moorestown, N.J.). Samples were imaged using a JEOL 6500F Scanning
Electron Microscope (JEOL, USA Inc., Peabody, Mass.).
[0075] Cytocompatibility and cell viability studies. All cell
culture materials, not otherwise specified, were purchased from
Gibco, Life Technologies. NIH-3T3 fibroblast cells were cultured in
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal
Bovine Serum (FBS), 2% L-glutamine (200 mM), 1% sodium pyruvate
(100 mM), and 1% pen-strep solution (5000 units/mL of penicillin
and 5 mg/mL of streptomycin, Sigma-Aldrich, St. Louis, Mo.). Cells
were maintained at 37.degree. C. and 5% CO.sub.2 with humidity and
split when reaching 80%-90% confluency by treatment with 0.25%
trypsin-EDTA and neutralization with complete media. Cells were
centrifuged for 6 min at 1600 rpm, then the cell pellet was
dispersed in HBSS, counted and adjusted to the required
concentration. 3D cell culture in K.sub.2 hydrogel was achieved by
mixing a 2% by weight peptide stock solution in 298 mM sucrose with
a cell suspension of 2.times.10.sup.6 cells/mL in HBSS. The final
concentration was 1% by weight K.sub.2 hydrogel in 149 mM sucrose
and 0.5.times.HBSS with 1.times.10.sup.6 cells/mL. For O.sub.5
peptide, hydrogel was prepared by sonication using only 90% of the
required 149 mM sucrose-0.5.times.HBSS to obtain a 1% by weight
concentration, the remaining 10% was added as a cell suspension,
and mixed by pipetting into the gel to obtain a final concentration
of 1.times.10.sup.6 cells/mL evenly distributed though the volume
of the hydrogel. 70 .mu.L of seeded hydrogels were cultured in a
Lab-Tek 16-well glass chamber (0.4 mm.sup.2, Thermo Fisher,
Rochester, N.Y.) with 200 .mu.L of medium and fed every 2 days by
replacing 100 .mu.L with fresh media. Cell viability was analyzed
using a live/dead viability kit (Invitrogen, Thermo Fisher,
Carlsbad, Calif.). At specific time points, the medium was removed,
and gels were rinsed with 1.times.PBS, then 100 .mu.L of staining
solution containing 2 .mu.M Calcein AM and 4 .mu.M Ethidium
homodimer-1 in DPBS were added. Cells were incubated from 15 to 30
min and imaged using a Nikon A1 Rsi fluorescent confocal microscope
(Nikon Instruments, Tokyo, Japan). Three z-stack images of 100
.mu.m deep per sample were taken at 20.times. using a 488 nm green
channel and 561 nm red channel lasers. Images were processed with
NIS Element and live/dead cell counting was performed on Imaris
3D/4D Image Processing software (Bitplane, Concord, Mass.).
[0076] To evaluate the cell morphology, hydrogels were rinsed with
PBS and fixed with 10% buffered formalin for 30 min Cells were
treated with 0.5% Triton X for 10 min and 100 mM glycine for 10
min, followed by 10% Bovine Serum Albumin (BSA) in HBSS block
solution. Then cells were stained for filamentous actin with Alexa
Fluor.RTM. 488-Phalloidin and for the nuclei with ProLong.RTM. Gold
Antifade DAPI mounting media. Cell in the hydrogels were analyzed
by confocal microscopy using a 40.times. water objective and
processed as described.
[0077] In vivo compatibility studies. All experimental procedures
were approved by the Rice University Institutional Animal Care and
Use Committee (IACUC) and performed according to the Animal Welfare
Act and NIH guidelines. Female C57BL/6 mice from 8 to 12 weeks were
purchased from Charles River (Wilmington, Mass.). Mice were
injected with 4 implants of 100 .mu.L 1% by weight O.sub.5 hydrogel
in the dorsal subcutaneous space using a 26-gauge needle. At days
3, 7, and 14 mice were euthanized, and implants were extracted,
fixed with 10% neutral buffered formalin and processed into
paraffin blocks. Tissue was sectioned at 5 .mu.m thickness and
stained with hematoxylin and eosin, and Masson's trichome under
standard procedures. For fluorescent immunostaining, tissue
sections were deparaffinized, hydrated and antigens were retrieved
by boiling in sodium citrate buffer (10 mM sodium citrate, 0.05%
Tween 20 at pH 6.0). Tissues were permeabilized with 0.5% Triton X
and blocked with 1% BSA in PBS for 30 min. Tissues were incubated
overnight at 4.degree. C. with rat anti-Mouse F4/80 monoclonal
antibody (1:50, clone BM8, eBioscience.TM., Cat#14-4801-82). Then,
samples were incubated for 1 hour with goat anti-rat
AlexaFluor.RTM. 568 (1:500, Invitrogen), followed by mounting with
ProLong.RTM. Gold Antifade reagent with DAPI. Tissue slides were
analyzed by confocal fluorescent microscopy.
Example 1--O.sub.n(SL).sub.6O.sub.n Series Peptides
[0078] Previously, a class of self-assembling peptides, called
Multidomain Peptides (MDPs), has been studied (Dong et al., 2007).
These peptides are designed with an ABA motif, where the B domain
alternates hydrophilic and hydrophobic amino acids while the A
domain contains charged residues. The balance between charge
repulsion at the ends of the peptide and stabilizing hydrogen
bonding and hydrophobic packing in the core controls the extent of
self-assembly. In aqueous solution, the peptides typically adopt an
antiparallel sheet conformation and assemble into nanofibers driven
by the hydrophobic effect and hydrogen bond formation between
backbone amides. Fiber length and stability are modulated by pH or
the addition of multivalent ions, which screen the electrostatic
repulsion and shift the balance of supramolecular forces towards
assembly of nanofibers. Upon achieving sufficient length, these
nanofibers entangle and form a viscoelastic hydrogel (Dong et al.,
2007; Aulisa et al., 2009; Bakota et al., 2013; Moore &
Hartgerink, 2017). Following a similar rationale, the neutral MDP
design presented here is composed of the same amphiphilic core with
alternating hydrophilic and hydrophobic amino acids comprised by
the twelve amino acid sequence (SL).sub.6. This domain drives
intermolecular .beta.-sheet hydrogen bonding and hydrophobic
packing. However, this domain by itself yields insoluble nanofibers
which precipitate in solution. Instead of using charged termini to
improve solubility, the current design selects hydroxyproline (here
abbreviated as O) as non-ionic termini. Hydroxyproline provides the
necessary hydrophilicity with its hydroxyl side chain and, more
importantly, cannot participate in the N--H backbone hydrogen bond
donation required in the formation of .beta.-sheets. Additionally,
hydroxyproline repeats are predisposed to form polyproline type II
(PPII) helices, which can provide a bulky terminus to establish
molecular frustration by steric impediment to the growing peptide
nanofibers. To study the effect of oligo-hydroxyproline on the
self-assembly of nanofibers and subsequent hydrogelation, MDPs with
a variable number of hydroxyproline residues,
O.sub.n(SL).sub.6O.sub.n where n=1-6, were designed and synthesized
(FIG. 1, Table 2).
TABLE-US-00002 TABLE 2 O.sub.n(SL).sub.6O.sub.n series and control
peptides Molar Mono- conc. of SEQ isotopic Molecular 1 wt.% ID mass
weight solutions Name NO: Sequence (Da) (Da) (mM) O.sub.1 1
Ac-OSLSLSLSLSLSLO-CONH.sub.2 1485.83 1486.71 6.7 O.sub.2 2
Ac-OOSLSLSLSLSLSLOO-CONH.sub.2 1711.93 1712.94 5.8 O.sub.3 3
Ac-OOOSLSLSLSLSLSLOOO-CONH.sub.2 1938.04 1939.17 5.1 O.sub.4 4
Ac-OOOOSLSLSLSLSLSLOOOO- 2164.15 2165.41 4.6 CONH.sub.2 O.sub.5 5
Ac-OOOOOSLSLSLSLSLSLOOOOO- 2390.25 2391.64 4.2 CONH.sub.2 O.sub.6 6
Ac- 2616.35 2617.87 3.8 OOOOOOSLSLSLSLSLSLOOOOOO- CONH.sub.2
K.sub.2 7 Ac-KKSLSLSLSLSLSLKK-CONH.sub.2 1772.10 1773.17 5.6
E.sub.2 8 Ac-EESLSLSLSLSLSLEE-CONH.sub.2 1775.89 1776.94 5.6
(Hyp).sub.6 9 Ac-OOOOOO-CONH.sub.2 737.34 737.75 1.4
Example 2--Secondary Structure Characterization of
O.sub.n(SL).sub.6O.sub.n Series Peptides
[0079] From the series of O.sub.6(SL).sub.6O.sub.6 peptide,
O.sub.1-O.sub.4 were poorly soluble in water (<0.5 mg/mL) and
resulted in the formation of a heterogeneous suspension. In
contrast O.sub.5 and O.sub.6 were found to be soluble in water at 1
mg/mL. At 10 mg/mL, O.sub.5 forms a hydrogel while O.sub.6 is a
viscous solution. The ATR-FTIR spectra (FIG. 2A) of the dried
peptide films presented a peak between 1620-1630 cm.sup.-1, which
corresponds to the amide I band of a .beta.-sheet conformation, and
a weak peak around 1695 cm.sup.-1 indicating an antiparallel
component. These results indicate that the core .beta.-sheet
structure is not significantly disturbed by the
oligo-hydroxyproline termini in the dried samples. Poor solubility
of O.sub.1-O.sub.4 made the characterization across all peptides in
uniform solvent challenging, therefore 2-2-2-trifluoroethanol (TFE)
was often used as a solubilizing agent for sample preparation and
characterization. Circular dichroism (CD) spectroscopy of peptide
solutions in TFE (1% by weight, 7-4 mM) was performed (FIG. 2B). CD
of O.sub.1 corresponds to a canonical .beta.-sheet structure,
presenting minima at 218 nm and maxima at 197 nm. Peptides O.sub.2
through O.sub.6 display a combination of different secondary
structures. However, increasing numbers of hydroxyproline residues
resulted in a corresponding increased fraction of PPII secondary
structure as determined using a least square linear fitting method
(Tables 2 & 3). While the peptides also showed an
.alpha.-helical component, this is due to the propensity of TFE to
promote this conformation (Sonnichsen et al., 1992; Roccatano et
al., 2002). Together the IR and CD suggests the secondary structure
of this series of peptides is .beta.-sheet in the amphiphilic
(SL).sub.6 core while the O.sub.n termini in O.sub.2-O.sub.6 adopt
a PPII helical structure. This PPII helix becomes large enough to
mediate solubility at O.sub.5 and O.sub.6.
TABLE-US-00003 TABLE 3 Estimation of the secondary structure of O
series peptides in TFE at 25.degree. C. from circular dichroism
data using the least square linear method % % % Predominant Peptide
PPII.sup.[a] .beta.-sheet.sup.[a] .alpha.-helix.sup.[a]
nmrsd.sup.[b] Structure O.sub.1 0 100 0 2.7 .times. 10.sup.-15
.beta.-sheet O.sub.2 29.3 28.8 41.9 1.53 .alpha.-helix O.sub.3 38.4
25.2 36.4 0.39 PPII/.alpha.-helix O.sub.4 41.8 17 41.2 0.25
PPII/.alpha.-helix O.sub.5 50 9.5 40.5 0.08 PPII O.sub.6 48.5 11.4
40.2 0.05 PPII .sup.[a]Percentage of each structure was calculated
from the regression estimator coefficients (SI Table 4, FIG. 8).
Basis spectra for the determination of peptide secondary structure
were: O.sub.1 for .beta.-sheet, (Hyp).sub.6 for polyproline type
II, and K.sub.2 for .alpha.-helix (FIG. 7). .sup.[b]nmrsd is
normalized root square deviation.
TABLE-US-00004 TABLE 4 Regression estimator coefficients of the
analysis of peptide solutions in TFE by the least square linear
method (.beta.-sheet, PPII, .alpha.-helix) .beta.- .alpha.- Major
Peptide sheet PPII helix rms.sup.[a] nmrsd.sup.[b] contribution
O.sub.1 1 0 0 1.04 .times. 10.sup.-12 2.7 .times. 10.sup.-15
.beta.-sheet O.sub.2 0.59 0.60 0.86 36.31 1.53 .alpha.-helix
O.sub.3 0.36 0.55 0.52 19.24 0.39 .alpha.-helix - PPII O.sub.4 0.26
0.64 0.63 23.12 0.25 PPII - .alpha.-helix O.sub.6 0.15 0.64 0.53
5.78 0.05 PPII .sup.[a]rms: root mean square .sup.[b]nmrsd:
normalized root square deviation
Example 3--Nanostructure of O.sub.n(SL).sub.6O.sub.n Series
Peptides
[0080] Negative-stain TEM was used to analyze the nanostructure of
the O.sub.1-O.sub.6 peptide series. The very poor water solubility
of O.sub.1 and O.sub.2 resulted in dense clusters of nanofibers
along with amorphous aggregates in their TEM images with only rare
areas being dispersed enough to see the underlying nanofibrous
structure (FIG. 2C). However, the dispersion of the nanofibers
increases with the number of hydroxyproline residues, improving
their visualization. Also, the apparent fiber length increases with
greater numbers of hydroxyproline residues. While fibers from
O.sub.1-O.sub.4 appear mostly short and rigid, O.sub.5 and O.sub.6
nanofibers are significantly longer and display more curvature,
suggesting a more flexible fiber. TEM was also performed on
O.sub.1-O.sub.6, which had been initially dissolved in TFE to help
disperse the assembled nanofibers and revealed similar trends to
those formed only in water (FIG. 9). These data suggest that the
increase in hydroxyproline residues and, therefore, PPII content in
the peptide structure, modulate the solubility and nanostructure of
the fibers by increasing both the hydrophilicity and steric
frustration in the supramolecular assembly. Too short of an O.sub.n
termini and the peptides assemble into short, rigid, low solubility
fibers that form dense aggregates and precipitate. In contrast, too
long of an O.sub.n termini and the peptide assembles into long,
easily solubilized fibers, but with too few physical crosslinks to
support gelation. Only O.sub.5(SL).sub.6O.sub.5 has the right
balance of fiber length, rigidity, and solubility to form a
hydrogel. This peptide remains a hydrogel over a wide pH range
(3-11) as well as in deionized water (FIG. 3) and common buffers,
such as phosphate-buffered saline (10 mM phosphate, 137 mM NaCl)
(FIG. 10) and Hank's Balanced Salt Solution (HBSS). Similarly, the
secondary structure as examined by CD has only minor changes under
this range of conditions demonstrating the robustness of the
neutral MDP approach. In contrast, charged MDPs, such as
K.sub.2(SL).sub.6K.sub.2 and E.sub.2(SL).sub.6E.sub.2, have a
strong dependence on pH and ionic strength with gels only forming
at neutral pH or at elevated ionic strength (FIGS. 3 & 11), and
significant changes to secondary structure are observed across
these conditions that correlate to the ionization or shielding of
their charged residues.
Example 4--Hydrogelation of O.sub.5 Peptide
[0081] Hydrogel formation of previously designed MDPs is achieved
by the addition of multivalent ions, such as phosphate, to induce
gelation at neutral pH. This is a convenient media since these
buffers are also required for most cell culture work. To assess the
biological potential of the O.sub.n series of peptides,
hydrogelation was tested in media that supports cell culture (1% by
weight in 149 mM sucrose and 0.5.times.HBSS) and is commonly used
to test charged MDPs, such as K.sub.2(SL).sub.6K.sub.2 and
E.sub.2(SL).sub.6E.sub.2 (Moore & Hartgerink, 2017). Under
these conditions, O.sub.5 forms a self-supportive hydrogel after
dispersion by ultrasonication while O.sub.1-O.sub.4 remain
insoluble and O.sub.6 dissolves to form a viscous solution. This
suggests that O.sub.5 reaches a state of optimal fiber length and
entanglement to create a stable hydrogel. FIG. 4A shows the CD
spectra of aqueous 1 wt. % (4.2 mM) O.sub.5 peptide solution in 149
mM sucrose and 0.5.times.HBSS. SEM analysis of the resulting
hydrogel reveals a high-density nanofiber network (FIG. 4B). The
viscoelastic properties of the O.sub.5 hydrogel were analyzed by
oscillatory rheology, and a storage modulus (G') of 23.3.+-.8.7 Pa
and a loss modulus (G'') of 1.7.+-.0.5 Pa were found. The low G'
value compared to previously studied MDPs (Aulisa et al., 2009),
which are typically in the range of 100-400 Pa, indicates that this
material is a relatively compliant and flexible gel, which is also
confirmed by the frequency sweep (FIG. 12). The O.sub.5 peptide
hydrogel exhibits liquid behavior when high strain is applied,
while having a very rapid recovery of approximately 92% of its
initial storage modulus 1 minute after the strain is released (FIG.
4D), making this supramolecular material easily injectable because
of its shear thinning and shear recovery properties. In comparison,
charged MDPs typically show a slower shear recovery of
approximately 80% in the same time frame and require approximately
10 minutes to reach 90% recovery (Aulisa et al., 2009).
Example 5--Cytocompatibility of O.sub.5 Peptide Hydrogel
[0082] Former MDP hydrogels, particularly lysine-based peptides,
have shown great promise for their use as biomaterials in part
because of their cytocompatibility (Moore & Hartgerink, 2017).
To study the effect of using non-ionic O.sub.5 flanking domains on
cell compatibility, 3D culture of NIH-3T3 fibroblasts was performed
in O.sub.5 peptide hydrogel and compared to culture in the
positively charged K.sub.2 peptide. Cell viability was evaluated
using a Calcein-AM and Ethidium homodimer-1 test (FIG. 5). At day
1, approximately 20% of cells are viable in K.sub.2 hydrogel,
whereas for O.sub.5 hydrogel, almost 70% survive after
encapsulation (FIGS. 5A-D). At day 3, these levels are relatively
unchanged. By day 5, fibroblasts are growing and reproducing in the
positively charged K.sub.2 hydrogel, achieving a similar percentage
of viable cells as O.sub.5 hydrogel. Overall, K.sub.2 is observed
to be somewhat cytotoxic during the first days of cell culture,
likely due to the high density of positively charged amines.
However, cells recover and can attach and proliferate within the
gel, and by day 5 cell density is significantly higher than days 1
and 3 (FIG. 5E). On the other hand, fibroblasts encapsulated in the
O.sub.5 hydrogel show good initial viability, which remains
constant throughout the experiment (FIG. 5D). Despite high and
consistent cell viability in O.sub.5 over the course of this study,
the encapsulated fibroblasts are not observed to proliferate. Cells
encapsulated in O.sub.5 gel do not spread and adhere to the
material like in K.sub.2, but instead they remain "balled up" as
shown in FIGS. 5B-C. Generally, this balled-up morphology
represents imminent cell death or apoptosis (Saraste & Pulkki,
2000), where cells die from 12 to 24 h after initiating this
process (Saraste, 1999; Messam & Pittman, 1998). Yet, cells in
the neutral MDP preserve this morphology through day 5 (120 h) and
show green fluorescence derived from Calcein AM, indicating an
intact membrane and cell survival. Cultured cells displaying this
balled-up morphology recover, grow, and proliferate when extracted
from the O.sub.5 peptide hydrogel after 5 days of culture (FIG.
13). All these observations suggest that fibroblasts cultured in
O.sub.5 may be induced to enter a quiescent state when encapsulated
within the hydrogel. The ability of O.sub.5 hydrogel to keep cells
alive without proliferation is a promising result with potential
applications for the use of this material in cell preservation. The
differences in cell viability, morphology, and proliferation are
all indicators of the substantial differences induced by fibers
with similar morphology but with different charge.
Example 6--In Vivo Host Response to O.sub.5 Peptide Hydrogel
[0083] To investigate the application of this peptide-based
material in regenerative medicine, the host response to O.sub.5
peptide hydrogel was evaluated in mice using a subcutaneous
injection model. The injected hydrogel boluses were examined at 3,
7, and 14 days after injection. FIGS. 14A-C shows histological
sections of the implants stained with Hematoxylin & Eosin and
fluorescent immunostaining for F4/80+ macrophages. At day 3 (FIG.
14A), F4/80+ macrophages have infiltrated the periphery of the
material, while some are migrating to the core. By day 7 (FIG.
14B), macrophages have completely infiltrated the implant. A
decrease in the implant size suggests that the O.sub.5 hydrogel is
degrading (FIGS. 15A-C). By day 14 (FIG. 14C) the hydrogel is
almost completely degraded, with fewer macrophages present.
Additionally, natural collagen is observed as the material is
remodeled into natural tissue (FIGS. 16A-C). Compared to K.sub.2
hydrogel, the neutral MDP O.sub.5 elicited a weaker inflammatory
response and did not promote vascularization or innervation (Moore
et al., 2018). However, in contrast with other neutral polymers,
such as PEG, the O.sub.5 hydrogel promotes rapid cellular
infiltration, is fully degradable and is not fibrously encapsulated
(Lynn et al., 2009), demonstrating that it has achieved some of the
"stealth" characteristics associated with PEG materials while also
maintaining the ability to interact with living systems.
[0084] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0085] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0086] U.S. Pat. No. 5,004,681 [0087] U.S. Pat. No. 8,099,297
[0088] U.S. Pat. Appln. Publn. 2009/0305325 [0089] U.S. Pat. Appln.
Publn. 2005/0106554 [0090] U.S. Pat. Appln. Publn. 2005/0277107
[0091] U.S. Pat. Appln. Publn. 2017/0335287 [0092] Aggeli et al.,
J. Am. Chem. Soc. 2003, 125, 9619-9628. [0093] Aulisa et al.,
Biomacromolecules 2009, 10, 2694-2698. [0094] Bakota et al.,
Biomacromolecules 2013, 14, 1370-1378. [0095] Bankwell et al., Nat.
Mater. 2009, 8, 596-600. [0096] Blau, Curr. Opin. Colloid Interface
Sci. 2013, 18, 481-492. [0097] Chockalingam et al., Protein Eng.
Des. Sel. 2007, 20, 155-161. [0098] Collier et al., J. Am. Chem.
Soc. 2001, 123, 9463-9464. [0099] Cormier et al., ACS Nano 2013, 7,
7562-7572. [0100] Dong et al., J. Am. Chem. Soc. 2007, 129,
12468-12472. [0101] Fischer et al., Biomaterials 2003, 24,
1121-1131. [0102] Habibi et al., Nano Today 2016, 11, 41-60. [0103]
Hanna & Hubel, Preservation of Stem Cells. Organogenesis 2009,
5(3), 134-137. [0104] Hartgerink et al., J. Am. Chem. Soc. 1996,
118, 43-50. [0105] Hartgerink et al., Science 2001, 294, 1684-1688.
[0106] Holmes et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
6728-6733. [0107] Hunt, Transfus. Med. Hemotherapy 2011, 38(2),
107-123. [0108] Kadlecova et al., Biomacromolecules 2012, 13,
3127-3137. [0109] Leach et al., Biomaterials 2018, 163, 67-75.
[0110] Li et al., Chem. Rev. 2015, 115, 8564-8608. [0111] Li &
Hartgerink, J. Am. Chem. Soc. 2017, 139, 8044-8050. [0112] Lutolf
& Hubbell, Nat. Biotechnol. 2005, 23, 47-55. [0113] Lynn et
al., J. Biomed. Mater. Res. Part A 2009, 93A, 941-953. [0114] Mazia
et al., J. Cell 1975, 66, 198-200. [0115] Menges, 2017, Version
1.2.7. [0116] Messam & Pittman, Exp. Cell Res. 1998, 238,
389-398. [0117] Meyers & Grinstaff, Chem. Rev. 2012, 112,
1615-1632. [0118] Micklitsch et al., Angew. Chemie--Int. Ed. 2011,
50, 1577-1579. [0119] Moore et al., Biomaterials 2018, 161,
154-163. [0120] Moore & Hartgerink, Acc. Chem. Res. 2017, 50,
714-722. [0121] Myers et al., Protein Sci. 1998, 7, 383-388. [0122]
Nisbet & Williams, Biointerphases 2012, 7, 1-14. [0123] Pochan
et al., J. Am. Chem. Soc. 2003, 125, 11802-11803. [0124] Powers et
al., Angew. Chemie-International Ed. 2002, 41, 127-130. [0125]
Rad-Malekshahi et al., Bioconjug. Chem. 2016, 27, 3-18. [0126]
Roccatano et al., Proc. Natl. Acad. Sci. 2002, 99, 12179-12184.
[0127] Saha & Jaenisch, Cell Stem Cell 2009, 5(6), 584-595.
[0128] Saraste, Herz 1999, 24, 189-195. [0129] Saraste &
Pulkki, Cardiovasc. Res. 2000, 45, 528-537. [0130] Schneider et
al., J. Am. Chem. Soc. 2002, 124, 15030-15037. [0131] Sonnichsen et
al., Biochemistry 1992, 31, 8790-8798. [0132] Stathopulos et al.,
Protein Sci. 2004, 13, 3017-3027. [0133] Stupp et al., Science
1997, 276, 384-389. [0134] Veiga et al., Biomaterials 2012, 33,
8907-8916. [0135] Webber et al., Nat. Mater. 2015, 15, 13-26.
[0136] Zhu, Biomaterials 2010, 31, 4639-4656.
Sequence CWU 1
1
20114PRTArtificial SequenceSynthetic polypeptideMOD_RES(1)..(1)Xaa
represents hydroxyprolineMOD_RES(14)..(14)Xaa represents
hydroxyproline 1Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu
Xaa1 5 10216PRTArtificial SequenceSynthetic
polypeptideMOD_RES(1)..(2)Xaa represents
hydroxyprolineMOD_RES(15)..(16)Xaa represents hydroxyproline 2Xaa
Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu Xaa Xaa1 5 10
15318PRTArtificial SequenceSynthetic polypeptideMOD_RES(1)..(3)Xaa
represents hydroxyprolineMOD_RES(16)..(18)Xaa represents
hydroxyproline 3Xaa Xaa Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu
Ser Leu Xaa1 5 10 15Xaa Xaa420PRTArtificial SequenceSynthetic
polypeptideMOD_RES(1)..(4)Xaa represents
hydroxyprolineMOD_RES(17)..(20)Xaa represents hydroxyproline 4Xaa
Xaa Xaa Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu1 5 10
15Xaa Xaa Xaa Xaa 20522PRTArtificial SequenceSynthetic
polypeptideMOD_RES(1)..(5)Xaa represents
hydroxyprolineMOD_RES(18)..(22)Xaa represents hydroxyproline 5Xaa
Xaa Xaa Xaa Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu Ser1 5 10
15Leu Xaa Xaa Xaa Xaa Xaa 20624PRTArtificial SequenceSynthetic
polypeptideMOD_RES(1)..(6)Xaa represents
hydroxyprolineMOD_RES(19)..(24)Xaa represents hydroxyproline 6Xaa
Xaa Xaa Xaa Xaa Xaa Ser Leu Ser Leu Ser Leu Ser Leu Ser Leu1 5 10
15Ser Leu Xaa Xaa Xaa Xaa Xaa Xaa 20716PRTArtificial
SequenceSynthetic polypeptide 7Lys Lys Ser Leu Ser Leu Ser Leu Ser
Leu Ser Leu Ser Leu Lys Lys1 5 10 15816PRTArtificial
SequenceSynthetic polypeptide 8Glu Glu Ser Leu Ser Leu Ser Leu Ser
Leu Ser Leu Ser Leu Glu Glu1 5 10 1596PRTArtificial
SequenceSynthetic polypeptideMOD_RES(1)..(6)Xaa represents
hydroxyproline 9Xaa Xaa Xaa Xaa Xaa Xaa1 5104PRTArtificial
SequenceSynthetic polypeptide 10Arg Gly Asp Ser1115PRTArtificial
SequenceSynthetic polypeptide 11Ile Lys Val Ala Val1
5123PRTArtificial SequenceSynthetic polypeptide 12Lys Asp
Ile11310PRTArtificial SequenceSynthetic polypeptide 13Arg Asn Ile
Ala Glu Ile Ile Lys Asp Ile1 5 10148PRTArtificial SequenceSynthetic
polypeptide 14Val Phe Asp Asn Phe Val Leu Lys1 5155PRTArtificial
SequenceSynthetic polypeptide 15Asp Trp Ile Val Ala1
51616PRTArtificial SequenceSynthetic polypeptide 16Ala Gln Phe His
Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Ala1 5 10
15179PRTArtificial SequenceSynthetic polypeptide 17Arg Glu Leu Arg
Tyr Leu Arg Arg Ala1 5187PRTArtificial SequenceSynthetic
polypeptide 18Phe Leu Pro Ala Ser Gly Leu1 5197PRTArtificial
SequenceSynthetic polypeptide 19Glu Ser Pro Leu Lys Arg Gln1
52011PRTArtificial SequenceSynthetic polypeptide 20Asp Pro His Ile
Lys Leu Gln Leu Gln Ala Glu1 5 10
* * * * *